Adenylyl cyclase 2 expression and function in neurological diseases

Abstract Adenylyl cyclases (Adcys) catalyze the formation of cAMP, a secondary messenger essential for cell survival and neurotransmission pathways in the CNS. Adcy2, one of ten Adcy isoforms, is highly expressed in the CNS. Abnormal Adcy2 expression and mutations have been reported in various neurological disorders in both rodents and humans. However, due to the lack of genetic tools, loss‐of‐function studies of Adcy2 are scarce. In this review, we summarize recent findings on Adcy2 expression and function in neurological diseases. Specifically, we first introduce the biochemistry, structure, and function of Adcy2 briefly. Next, the expression and association of Adcy2 in human patients and rodent models of neurodegenerative diseases (Alzheimer's disease and Parkinson's disease), psychiatric disorders (Tourette syndrome, schizophrenia, and bipolar disorder), and other neurological conditions (stress‐associated disorders, stroke, epilepsy, and Lesch‐Nyhan Syndrome) are elaborated. Furthermore, we discuss the pros and cons of current studies as well as key questions that need to be answered in the future. We hope to provide a focused review on Adcy2 that promotes future research in the field.


| INTRODUC TI ON
Adenylyl cyclases (Adcys) are a family of enzymes that catalyze the formation of cyclic adenosine 3′,5′-monophosphate (cAMP) from ATP. 1 cAMP is a vital second messenger that can act either in a kinase-dependent manner via protein kinase A (PKA) or in a kinase-independent manner via exchange protein directly activated by cAMP (Epac). 2 Each Adcy consists of two transmembrane helical regions (M 1 and M 2 ) and two cytoplasmic regions (C 1 and C 2 ), which are further divided into C 1a , C 1b , C 2a , and C 2b (Figure 1).These cytoplasmic regions contain the catalytic and dimerization domains. 3mmalian Adcys are grouped into five families based on their structure, function, and regulatory properties (Table 1). 1 Most can be regulated by G-protein-coupled receptors (GPCRs) via interactions with their stimulatory (G αs ) and inhibitory (G αi/o ) G α and/or dimerized G βɣ subunits. 4Group I Adcys (Adcy1, 3, and 8) are stimulated by Ca 2+ /calmodulin (CaM) and G αs , but inhibited by G αi/o and G βɣ .Group II Adcys (Adcy2, 4, and 7) are stimulated by G αs and G βɣ , but insensitive to Ca 2+ /CaM.Group III Adcys (Adcy5 and 6) are stimulated by G αs and G βɣ but inhibited by G αi/o and Ca 2+ .Group IV has only one member (Adcy9) and is activated by G αs , inhibited by calcineurin and PKC, and insensitive to forskolin. 1,5[27][28] Adcy2 catalyzes the production of cAMP, which acts indiscriminately on PKA and Epac to influence a multitude of cellular processes including adhesion, movement, and differentiation. 20][31][32][33] Adcy2 has also been reported as an NAD-binding protein, a family of proteins associated with multiple neurodegenerative disease-related pathways. 34 a member of the group II Adcys, Adcy2 is insensitive to Ca 2+ / CaM, but can be stimulated by G αs & G βɣ .It has been shown that Adcy2 can be regulated by phosphodiesterases (PDEs) and A-kinase anchoring proteins (AKAPs). 35For example, AKAP9, also known as Yotiao, directly inhibits Adcy2 via the N-terminus, increasing cAMP signal specificity. 36

| Alzheimer's disease
Alzheimer's disease (AD) is the largest cause of dementia in the U.S., affecting approximately 6.7 million individuals over 65 years of age in 2023. 37AD is characterized by the presence of extracellular amyloidβ (Aβ) plaques and intracellular tau tangles spreading from the hippocampus to the cortex in a slow propagative manner. 38,39[41] Studies have linked Adcy2 to AD and AD comorbidities.been found in OXYS rats, an inbred strain with an accelerated aging phenotype that spontaneously develops AD-like pathology, including cognitive deficits, hippocampal neuronal degeneration, oxidative and mitochondrial stress, and Aβ accumulation. 42Specifically, the number of hippocampal neurons decreases rapidly with age in OXYS rats, and this hippocampal neurodegeneration coincides with the downregulation of Adcy2. 42It should be noted that Adcy2 mRNA levels are downregulated in both Wistar control and OXYS brains at 5-18 months compared to the juvenile age. 43It remains unclear whether Adcy2 expression is further reduced in OXYS brains at 5-18 months compared to age-matched controls.Thus, it is possible that reduced Adcy2 expression is a consequence of aging rather than AD pathology.Direct evidence linking Adcy2 reduction to AD pathogenesis is lacking.Currently, it is unknown how Adcy2 level alters in other mouse models of AD, such as the 5xFAD model of Aβ pathology and PS19 model of tauopathy.
On the other hand, there are also studies showing a positive correlation between Adcy2 and AD.For example, a microarray dataset reports that Adcy2 is upregulated in the hippocampus of patients with severe AD (mean Braak stage 5.9). 44,45This direct evidence highlights a possible role of Adcy2 in AD pathogenesis.Given the role of cAMP in chemoattractant signaling and leukocyte extravasation in the CNS, increased Adcy2 in the hippocampus may indicate neurons in distress signaling for immune support. 46,47cy2 has been identified as a risk gene with a high number of single-nucleotide polymorphisms (SNPs) between AD and cognitive normal controls. 14,48These SNPs may affect Adcy2 functions, including vascular smooth muscle contraction, gap junction function, purine metabolism, chemokine signaling, and calcium signaling pathways, all of which become dysfunctional in AD. 49 The functional significance of these SNPs in AD pathogenesis needs future studies.
Besides SNPs, decreased Adcy2 methylation is found in AD and mild cognitive impairment (MCI) patients compared to healthy controls, with the greatest effects observed in males. 50Decreased methylation of Adcy2 may contribute to the previously discussed upregulation of hippocampal Adcy2 in AD patients.It should be noted, however, that despite the upregulation of hippocampal Adcy2, cAMP response element binding protein (CREB), the transcription factor that regulates genes involved in learning and memory, is impaired in AD patients. 51What causes the increased expression of Adcy2 is currently unknown.
Although there is no direct evidence linking Adcy2 to Aβ accumulation, Adcy2 is associated with amyloid precursor protein (APP) under acute stress conditions.It has been shown that inhibiting Adcy2 expression following acute stress reduces APP levels in DBA/2 J mice, 52 an inbred strain often used in aging and sensorineural studies.Subsequent studies show that cAMP derived from forskolin-sensitive Adcys, including Adcy2, stimulates APP production in neurons and astrocytes via adrenergic receptor activation. 53,54As a risk factor for dementia, chronic stress may increase Adcy2 expression and cAMP signaling, leading to elevated APP levels and consequently Aβ accumulation. 55However, a direct association between Adcy2 and stress preceding dementia has not been established.
Cerebral amyloid angiopathy (CAA), a risk factor for dementia and comorbidity of AD, is characterized by deposition of Aβ into cerebral blood vessels and decreased vessel integrity. 56Enrichment of Adcy2 is also observed in humans with spontaneous CAA. 57This indirect evidence suggests that Adcy2 may be involved in cerebrovascular pathology in AD brains.Echoing this finding, cAMP is increased in cerebral microvessels of AD patients compared to non-demented elderly controls, whereas comparable cAMP levels are observed in young and old wildtype rats. 58These results suggest that cAMP upregulation is caused by AD pathology rather than normal aging.

| Parkinson's disease
Parkinson's disease (PD), first described in 1817, is a neurodegenerative disorder characterized by motor defects such as tremor and rigidity. 59 Although the functional significance of Adcy2 in PD pathogenesis remains largely unknown, a negative correlation between Adcy2 and PD has been reported.First, Adcy2 is downregulated in the striatum of mice treated with 1-methyl-4-phenyl-1,2,3,6-te trahydropyridine (MPTP), a widely used mouse model of PD. 60 It should be noted that the downregulation of Adcy2 in PD brains may be simply due to the loss of dopaminergic neurons, since dopamine can activate cAMP signaling through dopamine receptors. 61Next, in humans, Adcy2 has been identified as a gene of interest in several key signaling pathways whose dysfunction increases PD risk, such as calcium signaling and glutaminergic synapse. 62,63In addition, PANTHER pathway analysis indicates that downregulation of Adcy2 and other genes may be responsible for the altered serotonin degradation and dopamine signaling in the subventricular zone of PD patients. 64 a recent GWAS, an SNP in Adcy2 was identified as a genetic factor that makes PD patients susceptible to levodopa-induced dyskinesia (LID). 65LID presents as abnormal movements such as stereotypic, choreiform, and throwing movements as well as dystonia that mainly involves the head, face, limbs, and trunk.It has been found that neuronal knockdown of Adcy2 resolves LID in a Drosophila model of PD by suppressing dopamine-like receptor 1 (D1). 65However, in a rat model of PD with chronic levodopa administration, Adcy2 is downregulated compared to acute and untreated controls. 66In the rat model, the reduction in Adcy2 may represent a compensation mechanism.This aligns with a theory that precise regulation of cAMP turnover in the striatum is necessary to prevent abnormal movement. 65,67Consistent with this, both gain-and loss-of-function mutations in Adcys cause imbalances in cellular cAMP levels and contribute to movement disorders. 65,68The previously mentioned SNP in Adcy2 may enhance its activity and cause overactive cAMP signaling in motor pathways, similar to mutations in the cytoplasmic region of Adcy5 in familial dyskinesia, which include gain-and loss-of-function mutations with regional severity. 68Mutations in the regulatory domains result in less severe symptoms and those in the catalytic domains more severe. 68Increased D1 signaling as a result of levodopa administration causes increased Adcy activity, and in the case of Adcy2, this may result in overstimulation of the enzyme with already enhanced function.

| Tourette syndrome
Tourette syndrome (TS) is a chronic neurodevelopmental disorder characterized by uncontrollable motor and vocal tics.TS has a childhood onset between 4 and 8 years of age, and tics generally improve or resolve in adulthood. 69,70Little is known about the pathophysiology of TS, but differences in axonal pruning during development and dysfunction in the reward and sensorimotor circuits of the striatum are thought to contribute. 69TS is also commonly comorbid with other disorders, including obsessivecompulsive disorder (OCD) and attention-deficit hyperactivity disorder (ADHD). 69,70rrent studies suggest that Adcy2 may play a dual role in TS.On one hand, there is evidence supporting a beneficial role of Adcy2.Loss of an intron/exon splice site in Adcy2 and recurrent deletion of a subunit of cAMP-activated PKA have been identified in TS. 15,71 These mutations reduce cAMP supply to neurons, which in turn prevents retrograde mitochondrial transport.In addition, they also decrease PKA signaling, which downregulates mitochondrial fission. 15Given the important functions of mitochondrial transport and dynamics in neuronal function, 15,72 it is reasonable to speculate that restoring Adcy2/cAMP signaling exerts a beneficial role in TS. 71 There are also studies reporting a detrimental role of Adcy2.
While hyperdopaminergic cortico-striatal activity was previously thought to underlie TS etiology, newer evidence instead suggests that inhibited dopaminergic signaling drives TS pathology. 69,73,74imulation of D2 receptors, which inhibits Adcy function and downregulates cAMP production, 75 has been found to relieve tics in both child and adult patients, 73,74 highlighting a detrimental role of Adcy2/cAMP in TS.
One possible explanation for the distinct roles of Adcy2 in TS is its subcellular location.Mitochondria in neurons are mainly found in the axons, whereas D2 receptors are predominantly found at the synapses.It is likely that Adcy2 may exert a beneficial role in axons, but a detrimental role in synapses.The exact mechanism underlying the dual role of Adcy2 in TS needs further investigation.

| Schizophrenia
Schizophrenia (SZ) is a psychiatric disorder with positive symptoms (e.g.delusions and audio-visual hallucinations), negative symptoms (e.g.lack of emotionality), and cognitive decline.Impairment in social interactions and self-care often precede the negative or positive symptoms. 76Developmental and early childhood conditions, decreased synaptic pruning, and genetic variants are all thought to contribute to SZ. 76,77 In a rat model of SZ, reduced Adcy2 expression has been found in various brain regions.Specifically, lower levels of Adcy2 are detected in the nucleus accumbens (NAc), an important structure that coordinates emotional reaction, and the prefrontal cortex of juvenile SZ rats. 78In adult SZ rats, such reduction of Adcy2 is only observed in the NAc. 78Since Adcy2 is a downstream target of dopaminergic and glutamatergic signaling in the NAc, its reduction may contribute to the negative symptoms of SZ by downregulating emotional responsiveness. 79,80cently, an SNP (rs58502974) in Adcy2 has been identified as a susceptibility factor for SZ in an Iranian population with the AA allele being highly associated with SZ, while the T allele is protective. 16In addition, AKAP9, a direct inhibitor of Adcy2 that regulates cAMP signal specificity, 36 has been linked to SZ in a recent GWAS. 81Specifically, the K873R single-nucleotide variation (SNV) on AKAP9, which has a possibly damaging role, has been identified in 4 out of 572 SZ cases. 81It remains unclear how this SNV, which occurs in the Adcy2 binding region, affects the inhibitory effect of AKAP9 on Adcy2 activity. 81Since AKAP9 expression is highest in retinal photoreceptor cells, 82,83 it is speculated that decreased AKAP9 inhibition of cAMP in these cells may cause overactive phototransduction and regulation of light/dark specificity, contributing to the positive symptom of visual hallucinations. 84

| Bipolar disorder
Bipolar disorder (BD) is an inheritable chronic mood disorder with a high risk of suicide.Patients with BD have episodes of major depression or mania and often possess comorbidities of anxiety and substance abuse disorders. 85,86The high rate of heritability indicates a prominent genetic component.While multiple genes have been associated with BD via GWAS, none have been determined as causative or to carry major risk for BD thus far. 86owledge on Adcy2 in BD predominantly comes from human studies.One study shows that Adcy2 is associated with early-onset BD when dysfunction in serotonin and dopamine signaling arise. 87is association is also seen in the same pathways in ADHD. 87other report finds that Adcy2 is upregulated in both astrocytes and neurons in BD patients. 88In addition, a missense variant (rs13166360) in Adcy2 is associated with general BD risk, but some polymorphisms are associated with severity of the disease. 89For example, Iranian BD patients with the C allele of the rs2290910 polymorphism are more likely to suffer suicidal ideation, and those with the T allele are more likely to attempt suicide. 90Similarly, several other polymorphisms have been associated with the onset or incidence of BD in male patients in Chinese Han population. 91terestingly, there are no gender differences in the onset, severity, or symptoms of BD, 86,92 suggesting that while genetics may predispose an individual to BD, other factors also contribute to its pathogenesis.

| Stress-associated disorders
Multiple neurodegenerative and psychiatric conditions have been associated with stress, which acts as either a major causative or risk factor during disease development. 93,94Adcy2 has not been examined within the context of acute or chronic stress models, but differential gene expression has been observed in stress-associated disorders.
Stress has been identified as a risk factor for dementias, including AD. Human data shows that exposure to stress early in life is correlated with a higher risk of dementia. 55,95Although Adcy2 is associated with APP levels under acute stress conditions (see Alzheimer's Disease section), its role in stress remains largely unknown.
Anxiety and depression are common stress-associated disorders.One study found significantly decreased Adcy2 in the hippocampus and prefrontal cortex in young males of a mouse model of anxiety. 96Echoing this finding, ego network analysis identified Adcy2 as a top candidate gene related to anxiety disorders with the catechol-O-methyltransferase (Comt1) overexpression phenotype. 97Interestingly, Adcy2 is similarly dysregulated, among other retrograde endocannabinoid signaling pathway genes, in patients with major depressive disorder (MDD), although the directionality of gene expression is not stated in this study. 98bstance abuse disorders, including alcohol use disorder (AUD), are frequently comorbid with anxiety and stress disorders. 99,100It has been reported that Adcy2 is downregulated in the hippocampus of rats with an innate preference for alcohol. 101There are currently no data on a specific role of Adcy2 in AUD.However, given the important role of Adcy2 in regulating neurotransmitters in the NAc, a region heavily involved in the reward system and substance abuse disorders, it is reasonable to hypothesize that Adcy2 participates in the pathogenesis of AUD.

| Stroke
Stroke is one of the leading causes of death and disability worldwide.It is caused by either a lack of blood supply to the brain (ischemic stroke) or bleeding into the brain (hemorrhagic stroke). 102,103Although subtype-specific features exist, both ischemic stroke and hemorrhagic stroke share common pathologies including excitotoxicity, neuroinflammation, oxidative stress, and angiopathy. 103rrent studies on Adcy2 in stroke mainly come from ischemic stroke.In a GWAS, a gene locus associated with factor VII-activating protease (FSAP) activity (rs35510613) was identified 19 kb upstream of Adcy2 in ischemic stroke patients. 104,105Two SNPs (rs12652415 and rs1609428) located in an intron of Adcy2 in Swedish populations are thought to interact with rs35510613 to regulate FSAP activity. 105Interestingly, an in vitro study suggests that increased cAMP levels upregulate transcription of Habp2, the gene encoding FSAP, via PKA activation. 105Thus, Adcy2 may upregulate FSAP expression, and individuals with the rs12652415 and rs1609428 SNPs in Adcy2 may further enhance FSAP enzymatic activity.Subsequent loss-offunction studies show that genetic ablation of FSAP increases neointima and leukocyte recruitment following vascular injury in skeletal muscle in mice, whereas inhibition of FSAP improves BBB integrity and ischemic stroke outcomes in humans. 104,106Whether this difference is species (rodent vs. human)-and/or tissue (muscle vs. brain)dependent is currently unknown and should be determined in future research.
Currently, there are no studies on the expression and function of Adcy2 in hemorrhagic stroke.

| Epilepsy
Epilepsy is a disease where patients have multiple seizures within 24 hours, or the 10-year risk of seizure is >60% in affected individuals. 107Adcy2 mRNA is downregulated in the hippocampus of both individuals with temporal lobe epilepsy and mouse models of status epilepticus. 108Interestingly, a proteomics study reports upregulated hippocampal Cox2, an enzyme that stimulates Adcy2 activity, in a rat model of temporal lobe epilepsy. 109Consistent with this observation, cAMP is proconvulsive when injected into rodents' brains, 110,111 and a previous study found that a region of mouse chromosome 13 including Adcy2 contains a candidate gene(s) for audiogenic seizure development. 112The discrepancy between mRNA and protein expression in human data and model species may be caused by different detection approaches (mRNA vs. proteins).
Therefore, it is essential to investigate Adcy2 changes at both mRNA and protein levels in future research.

| Lesch-Nyhan syndrome
Lesch-Nyhan syndrome (LNS) is an X-linked developmental disorder characterized by dysfunctional purine metabolism, mental retardation, and uncontrollable self-injury. 113In the hypoxanthine phosphoribosyl transferase (HPRT)-deficient rat B103 neuroblastoma model of LNS, Adcy2 is almost completely absent, 114 indicating a possibly important role of Adcy2 in LNS.However, the relationship between Adcy2 and LNS needs to be validated in more sophisticated models (e.g. in vivo) in the future.

| CON CLUS I ON AND FUTURE DIREC TIONS
Adcy2, converting ATP to second messenger cAMP in cells, regulates multiple important functions.In the CNS, dysfunction in vivo impossible, which presents significant challenges in the field.Developing these genetic tools will enable in-depth functional studies and shed light on the significance of Adcy2 in neurological disorders.Third, is there functional compensation between Adcy2 and other Adcys?Given the existence of multiple Adcy isoforms, it is reasonable to believe there is functional redundancy.
However, the specific Adcy isoforms involved in compensation remain largely unknown, which relies on our understanding of Adcy expression in each cell type under both physiological and pathological conditions.Compound knockout mice, in which multiple Adcy isoforms are ablated, may be useful in determining functional redundancy in vivo.Fourth, is the function of Adcy2 completely dependent on its adenylyl cyclase activity?Although Adcy2 acts on ATP and converts it to cAMP, we cannot exclude the possibility that it has adenylyl cyclase-independent functions.
Adcy2 mutants lacking adenylyl cyclase activity may be used to answer this question.Last, it is essential to validate results from animal studies in human samples.There are multiple documented differences in Adcy2 expression between rodent models of disease and human patients.One possible reason is different methods and/or brain regions used in these studies.It should be noted that mRNA changes do not always echo alterations at the protein levels.Another possible reason is species difference.Rodents can replicate some, but not all, disease pathology in human patients.
Thus, key findings from animal research should always be validated in human cells or tissues.
Answers to these questions will enable a comprehensive understanding of the expression and function of Adcy2 in neurological diseases, which will substantially move the field forward.

| 3 of 11 GRAY
However, how exactly Adcy2 levels change in AD brains is controversial.On one hand, a negative correlation of Adcy2 with AD has F I G U R E 1 Adenylyl cyclase 2 (Adcy2) structure and classical signaling pathways.(A) Linearized Adcy2 structure with M1/M2 transmembrane domains highlighted.(B) Intermembrane Adcy2 structure with M1/M2 transmembrane domains (red), C1a/C2a catalytic sites (yellow), and C1b/C2b regulatory and dimerization domains (yellow).(C) Classical Adcy2 signaling pathways.G protein as well as PDEs and AKAP regulate the production of cAMP, which controls transcription via PKA and/or Epac/Rap1 activity.Rap1 can also interact with downstream effectors directly.Created with BioRender.et al.
Adcy2 mutations and dysregulation in neurological diseases.Adcy2 research.Many previous studies used indirect readout of Adcy2 activation, such as G αs /G βɣ dependence, which is not accurate since it can apply to other group II Adcys. 115Although Adcy2 global knockout mice have been generated and are being investigated currently, tissue-specific knockouts and inducible knockouts are not available.In addition, Adcy2 reporter mice and tissue-specific/inducible Adcy2 overexpression mouse lines are not available either.The lack of these genetic tools makes cellspecific and/or controllable manipulation of Adcy2 expression 114 Abbreviations: AD, Alzheimer's disease; AUD, alcohol use disorder; BD, bipolar disorder; CAA, cerebral amyloid angiopathy; HPC, hippocampus; LNS, Lesch-Nyhan syndrome; NAc, nucleus accumbens; PD, Parkinson's disease; PFC, prefrontal cortex; SVZ, subventricular zone; SZ, schizophrenia; TS, Tourette syndrome.a Mean age of subjects.b Males.c Median age of subjects.